Methods for separating constituents of biologic liquid mixtures

ABSTRACT

Centrifuges are useful to, among other things, remove red blood cells from whole blood and retain platelets and other factors in a reduced volume of plasma. Platelet rich plasma (PRP) can be obtained rapidly and is ready for immediate injection into the host. Embodiments may include valves, operated manually or automatically, to open ports that discharge the excess red blood cells and the excess plasma while retaining the platelets and other factors. High speeds used allow simple and small embodiments to be used at the patient&#39;s side during surgical procedures. The embodiments can also be used for the separation of liquids or slurries in other fields such as, for example, the separation of pigments or lubricants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional U.S. application Ser. No. 13/568,629,filed on Aug. 7, 2012, which application claims the benefit under 35U.S.C. §120 of application Ser. No. 12/949,781, filed on Nov. 19, 2010and entitled “Centrifuge” and the entire contents of both applicationsare expressly incorporated herein by reference thereto.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains to centrifuges.

2. Discussion of Related Art

Fluids, such as whole blood or various other biological fluids aresuspensions and can be separated into their constituent parts orfractions. For example, whole blood comprises four main fractions, redblood cells, white blood cells, platelets and plasma, that can beseparated based on their different specific gravities in a device suchas a centrifuge. An anti-coagulated whole blood sample may be placed ina test tube, or other similar device, which is then spun in a centrifugeat a specified speed. The generated centrifugal force separates theblood into the different fractions based on their relative specificgravities. The red blood cells are on the bottom, plasma, is on the topwith the intermediate specific gravity white blood cells and plateletsintermediate to the other two fractions. Various other biological fluidsmay be separated as well. For example, nucleated cells may be separatedand extracted from bone marrow or adipose tissue derived samples.

It is desirable to isolate the different fractions of whole blood fordiffering medicinal purposes. The platelets can be obtained inpreparations of platelet rich plasma (PRP) or platelet concentrates(PC). Platelets contain growth factors (e.g. PDGF, TGF-β, and others),which may initiate, aid in or accelerate various bodily functions,including but not limited to angiogenesis, wound healing, andosteogenesis. Administering autologous platelets to an injury site mayimprove the healing response by using a patient's own platelets withoutthe risk of infection by using blood products from another donor source.

Various systems exist for the production of PRP/PC. Some use specializedtest tubes, U.S. Pat. Nos. 7,179,391 and 7,520,402, that can includefloats, tubing and/or gel materials of specific densities. Other systemsuse specialized double syringes, for example those found in U.S. Pat.Nos. 6,716,187 and 7,195,606. These test tubes and syringes must becentrifuged in a specialized large centrifuge for a specified time,typically 10-30 minutes, and then by delicate handling and extraction ordecanting procedures produce the desired PRP/PC. The consistency ofthese preparations can vary depending on the operator's skill level.Other systems, for example U.S. Pat. No. 6,982,038, contain specializedcentrifuge chambers and complicated control systems to produce thePRP/PC in about 30 minutes. All of these systems provide PRP/PC ofdiffering platelet concentrations depending on the method used. A majordrawback to these methods is the need for an expensive piece of capitalequipment which limits the utility to facilities that have the funds andspace available. These methods also require considerable operator skillsto complete the procedures necessary to obtain the PRP/PC.

The ability to produce PRP/PC from a patient's own blood at the point ofcare without the need for complex, expensive equipment and difficultprocedures would facilitate the clinical utility of PRP/PC. Thereforethe objects of this invention include among other things providing anapparatus and method for processing a patient's own blood at the pointof care in a short period of time that is self contained, batteryoperated, small and or portable, inexpensive, easy to use, reproducible,able to separate many cellular populations, and disposable without theneed for additional centrifugation equipment

SUMMARY OF THE INVENTION

In accordance with the invention, a single use, sterile, self-contained,compact, easy to use centrifugal separation unit provides for quick,reliable platelet concentration from whole blood. The resultant PRP/PCcan be immediately used for application to the patient. The unit issuitable for office, operating room, emergency use, or military fieldhospital use.

The disposable self-contained PRP separator features a motor with adrive axis, the drive axis being coaxial with the central orlongitudinal axis of the blood separation chamber (BSC) assembly. Themotor can have the capacity to rotate the BSC at speeds in the range10,000 to 25,000 RPM for several minutes. Power can be supplied to themotor through a battery or other power pack. The power can be connectedthrough a switch and even small dry cell batteries will have sufficientcapacity to complete the separation process. The BSC and motor/batteryare fully enclosed in an outer container that includes an access port tothe BSC to which a standard syringe can be attached. Alternatively theBSC can be rotated by non-electrical means such as an air driven turbineor spring drive. It could also include a magnetic or mechanical couplingto an external drive motor, or any source of energy that may beavailable at the surgical site for example in the surgical suite or onlocation during a trauma procedure, such as at a “MASH” compound..

In a first embodiment the BSC assembly features a barrel that may becylindrical or tapered, an end cap incorporating passageways and atubular extension, and in some embodiments a piston or bladder, thatbetween them define the BSC. A sleeve sliding over the outer diameter ofthe end cap acts as the moving part of two valve assemblies, each valvefeaturing a recess in the outer surface of the end cap and an O-ring inthe recess. Passages within the end cap lead from the BSC to the recesscenters, and two ports in the sleeve align with the recess centers in a3 position sequence. The two ports in the sleeve are positioned so thatthey do not align with the two recess centers in the end cap at the sametime. In sequence the sleeve selects a first port open, then both portsclosed, and then a second port open. The ports are opened in a stepwisemotion, but could be opened proportionally. The sleeve is operated by aknob connected to a slidable collar through a bearing assembly so thatthe knob does not rotate during operation of the motor.

Anti-coagulated blood is injected through the tubular extension in orderto fill the BSC. The sleeve is in a first position where both ports onthe sleeve do not align with either of the recesses in the end cap. Themotor is actuated and the BSC rotates to create a centrifugal force onthe blood thereby separating it into its components with the red bloodcells closest to the inner wall of the BSC with the white blood cellslining the red blood cell layer toward the center, followed by theplatelets and then plasma filling the center. In other words, thecentrifugation yields concentric stratified constituent layers of themixture, with adjacent concentric stratified constituent layers defininga mixture interface. After a centrifugation period of about 1 minute orless the sleeve is moved to a second position in which the first port inthe sleeve aligns with the recess in the end cap. This port communicateswith the layer of red blood cells against the inner wall. The red bloodcells will exit the chamber through this port due to pressure generatedby the centrifugal force. As red blood cells exit the separator, thevolume is replaced by air entering through the tubular extension in theend cap. The air forms a column in the center of the chamber that growslarger as more volume is replaced. After a prescribed volume of redblood cells are discharged from the blood separator volume, the sleeveis moved to a third position to close the first port and open the secondport. This is done before the layer of platelets in the volume can exitthe first port. The passage to the second recess in the end cap of thedevice is precisely positioned away from the center axis to remove aprescribed volume of plasma from the BSC without disturbing the plateletlayer. As plasma leaves the chamber, air replaces the volume through thetubular extension and the column of air in the center of the BSCcontinues to grow in diameter. When the diameter of the air columnencompasses the second passage entrance, no more plasma can exit thechamber and the concentration process is thereby automatically ended.The device is turned off and the platelet concentrate is ready for use.

Another embodiment uses a flexible bladder lining the interior of theBSC. The solid end of the BSC includes a hole for air to enter aroundthe exterior of the flexible bladder. The end cap axis tubular extensionincludes an airtight valve. This embodiment operates in the same mannerexcept that it does not deliberately introduce air into contact with theblood sample. During the centrifugation cycle while red blood cells andthen plasma are exiting the chamber, air enters the opposite side of thechamber thus collapsing the flexible bladder. Due to the pressuregenerated in the liquid by centrifugal force, the sack collapses into a“W” shape with the open ends of the “W” facing toward the end of thechamber opposite the end with the air bleed hole. As more plasma exitsthe chamber the middle of the “W” reaches the second passage in the endcap and closes the passage off thus automatically ending the cycle.

Another embodiment replaces the flexible bladder with a piston andspring: as red blood cells (RBCS) exit the valve ports, the piston movestowards the end cap encouraged by the spring.

It is further disclosed that the system of the subject invention mayincorporate an automatic shutoff mechanism to seal the port(s) basedupon certain conditions. For example, one such mechanism can incorporatea flowable separator gel of an intermediate specific gravity selected tobe between an undesired element, e.g. red blood cells, and a desiredtherapeutic element, e.g. platelets. The separator gel viscosity isdesigned so that it will not pass through the small exit port at thecentrifuge speed employed in the blood separation centrifuge. Uponactivation of the centrifuge, the separator gel would create a distinctlayer and barrier between the outer red blood cell layer, located nearthe periphery of the axis of rotation, and the platelet poor layer whichwould be located closer to the center axis of the centrifuge rotation.The separator gel automatically plugs the first port when all of the redblood cells have exited. As a further example, the automatic shut-off ofthe first port can be accomplished with a solid damper, or vent flap,also constructed of a material with a specifically targeted intermediatespecific gravity. Upon initial operation, the damper would open andseparate away from the vent hole based upon its specific gravity andattempt to position itself at a location between the red blood cells andthe platelets. As in the previous example, once the red blood cells havefully exited the system, the damper would seal the vent hole andeffectively prevent the platelet rich fluid from exited the system. Asyet another example, plastic beads such as microspheres with the desiredintermediate specific gravity could also be pre-located within thecentrifuge chamber. The beads would be sized appropriately to plug theexit port after the undesirable element, e.g. red blood cells, exitedthe system.

In another embodiment, the BSC can be made of a clear (transparent)material so that the progress of the red blood cell removal can beobserved through a clear window in the outer case. This can allow forprecise timing for closing the first port to end the exiting of the redblood cells.

Another embodiment accomplishes the concentration through precise timingof the valve opening/closing sequence and the starting and stopping ofthe motor.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1 a and 1 b: Principle of operation

FIG. 2: Centrifuge with spring loaded piston in tapered chamber, chargeposition, RBC valve open, Plasma valve closed (Longitudinal partsection)

FIGS. 3 a, 3 b, 3 c, and 3 d show transverse sections of the centrifugewith spring loaded piston in tapered chamber, (transverse sections ofFIG. 2), and enlarged details of the RBC valve components used in alldevices shown in FIGS. 2, 4, 5, 6, 7, 9,10,11, 12, 14, 15, 16, 17, and18.

FIG. 4: Centrifuge with spring-loaded piston in tapered chamber,spin-down, RBCs separated from plasma, both valves closed (Longitudinalpart section).

FIG. 5: Centrifuge with spring-loaded piston in tapered chamber, midposition, RBC valve open and RBCs being dumped, plasma valve closed(Longitudinal part section).

FIG. 6: Centrifuge with spring-loaded piston in tapered chamber, finalposition, RBC valve closed, plasma valve open and most of plasma dumped(Longitudinal part section).

FIG. 7: Centrifuge with bladder chamber, charge position, RBC valveopen, plasma valve closed (Longitudinal part section).

FIG. 8: Centrifuge with bladder chamber, charge position, (transversesection of FIG. 7.)

FIG. 9: Centrifuge with bladder chamber, spin-down, RBCs separated fromplasma, both valves closed, (longitudinal part section).

FIG. 10: Centrifuge with bladder chamber, RBCs dumping position, RBCvalve open, plasma valve closed (Longitudinal part section)

FIG. 11: Centrifuge with bladder chamber, Plasma valve open, RBC valveclosed, plasma being dumped (Longitudinal part section)

FIG. 12: Centrifuge with air core, initial charge position, both valvesclosed. (Longitudinal part section)

FIG. 13: Centrifuge with air core, (transverse section of Fig. 12.)

FIG. 14: Centrifuge with air core, spin and separate, RBCs being dumped,RBC valve open, plasma valve closed (Longitudinal part section)

FIG. 15: Centrifuge with air core, RBC valve closed, plasma valve open,residual RBCs and residual plasma remaining (Longitudinal part section)

FIG. 16: Centrifuge with air core, removal of PRP at finish, both valvesclosed (Longitudinal part section)

FIG. 17: Centrifuge with a typical enclosure (Longitudinal part section,showing RBC and plasma capture means and aerosol prevention means)

FIGS. 18 a and 18 b: Centrifuge with typical enclosure, (transversesection of FIG. 17)

DETAILED DESCRIPTIONS OF THE EMBODIMENTS OF THE INVENTION

FIG. 1 a provides an illustration for description of the principle ofoperation of the devices covered in this invention. A chamber ofessentially frusto-conical shape 1, contains a mixture of severalliquids of differing specific gravities, and rotates about thelongitudinal axis XX. The liquids 2, 3, and 4 separate into radiallydistinct layers as shown in section AA. The taper is beneficial inseveral ways, first it allows a small volume of liquid to offer a largeradial depth (as shown at 11) compared with the radial depth the samevolume would have if distributed over the whole length of a rightcircular cylinder of similar dimensions, see FIG. 1 b at 14. Second, thetaper provides a component of radial acceleration force that helps toscour the outer liquid constituent towards a port 9 placed at the largercone diameter. Third, the taper also allows visualization of theconstituent boundaries as axial locations such as 5 and 6 instead ofradial locations such as 7 and 8 in some of the embodiments. In severalembodiments the wall 12 of FIG. 1 moves toward the larger diameter andthe frusto-conical volume reduces as one or more constituents are portedfrom the ports, for example at 9 and 10, leaving the center constituent3 at its original volume. In other embodiments wall 12 remains in placeand air is introduced on the center line at 13 to permit the porting ofconstituents 2 and 4 at 9 and 10 as the air core expands to replace thedischarged constituents.

FIG. 2 is a mainly longitudinal section of an essentially circulardevice, external housing not shown. In FIG. 2 a liquid tight variablevolume, the chamber (BSC), is formed from a tapered barrel 206, piston210, piston seal 211 and end cap 215. Piston 210 and seal 211 are biasedtoward the larger end of the BSC by spring 209. Larger end of barrel 206is closed by end cap 215. The inner surface of the end cap 215 forms thelarger diameter end wall of the chamber, with the inner surface of thebarrel 206 forming the chamber's tapering side wall. In the case wherethis device is used to enrich plasma from whole blood, end cap 215 haspassages 216 and 217 bored within to permit the passage of red bloodcells from passage 217 and plasma from passage 216. Passage 217 is shownpassing through the outside skirt of the end cap that is in line withthe outside wall of tapered barrel 206. A passage bored 90° from thatshown at 217; through the inside face of end cap 215 at the maximum IDposition would be functionally equivalent to the one shown at 217 andwould have a shape similar to passage 216. Passages 217 and 216 connectwith valves formed by O-rings 218 compressed in recesses 226 operatingin concert with ports 228 and 227 respectively in sleeve 213. Thesevalve components are shown enlarged in FIGS. 3 b and 3 d. Sleeve 213fits slidably on end cap 215 to permit the port holes 228 and 227 toconnect with the passages 216 and 217 at appropriate points in theoperation. Sleeve 213 is keyed to end cap 215 to permit the transmissionof rotary motion between these constituents (key not shown). Insert 219is fastened to end cap 215 to provide an axle for the ball bearing 220supporting the left hand end of the rotating assembly. Since the sleeve213 is rotating with the chamber, a ball bearing 221 is provide toconnect the sleeve to a non-revolving knob 223 via collar 225 and rods222. The knob and sleeve can be placed in 3 positions: first position,port 228 open and port 227 closed: second position, both ports 227 and228 closed: third position, port 228 closed and port 227 open. Barrel206 is fastened to the shaft 205 of electric motor 201 using screw 207.No additional bearings are provided at the motor end, the motor bearingssufficing to support the barrel. The complete assembly is supported by aframe 208, the insert bearing 220 and the motor 201 being located onthis same frame. The rotating components all rotate about axis XX.

To use the device for preparing PRP, a syringe 233 with needle 234,filled with anti-coagulated whole blood is inserted into the devicethrough elastomeric seal 214 to load the chamber with whole blood 229.Knob 223 is placed in the first position to allow air to discharge fromport 228 as the chamber is filled with blood. Whole blood 229 fullycharges the chamber pushing the piston 210 and seal 211 to the farright, compressing spring 209.

FIG. 3 a, a cross section at AA in FIG. 2, clarifies the construction ofthe knob 223 and rod components 222. FIG. 3 b is a cross section at BBin FIG. 2 showing details for the valve components, those being therecess 226 in end cap 215, O-ring 218 and port 228 in sleeve 213 (theconstruction of the valve for port 227 is the same). FIG. 3 c shows thesection at CC of FIG. 2.

Once the chamber has been charged with whole blood, the knob and sleeveare placed in the second position with both valves closed, the syringe223 is removed and the motor started. The motor is then run for timesbetween 15 and 90 seconds depending on the speed used. Speeds of 10,000rpm to 25,000 rpm have been used, developing centrifugal accelerationsat the outside of the spinning chamber from 1000 g to 6000 g.

FIG. 4 shows the device of FIG. 2 in operation rotating at speed. TheRBC port 228 and the plasma port 227 are both closed. The boundarybetween the RBC layer and the plasma layer is shown at 237. The piston210 is still at the as-charged position and the spring 209 is fullycompressed. The spring has two functions, it moves the piston to theleft as red blood cells are discharged from the chamber through port228, and the spring creates a significant minimum pressure in therevolving liquid: this prevents the core of the spinning liquid fromreaching the vapor pressure of the liquids and may suppress cell damagein some circumstances.

Once the red blood cells and the plasma have separated, with the devicestill rotating, the knob and sleeve are placed in the first position andred blood cells are discharged from port 228 into the casing (casing notshown, but see FIGS. 17 and 18) surrounding the device. FIG. 5 shows thesituation at the mid-point of the RBC 231 discharge when the piston 210is in mid position. Once the majority of red blood cells have beendischarged the valve is placed in the third position and plasma 230 iseliminated from port 227. FIG. 6 shows the situation at the end of theenrichment process: the plasma port 227 is still open and the piston isclose to the far left position: platelets that have a specific gravitybetween that of plasma and RBCs are trapped at the RBC-plasma boundarylayer 237; the plasma port is about to be closed and the motor stopped.

Typical volumes for the chamber are 20-100 mL, and the amount ofenriched plasma removed at the termination of the procedure isapproximately a quarter to an eighth of the original volume depending onthe degree of enrichment desired.

In order to retain all the platelets and other factors gathering at theRBC-plasma boundary, it is essential to close port 228 before all theRBCs have been removed, otherwise there is the danger of theseconstituents flowing out with the last RBCs. To ensure that this doesnot occur, the blood sample hematocrit value is used to judge theresidual volume of the chamber when the RBC port must be closed. Thisvolume is observable as a piston axial position, and the valve is movedfrom position one to position three as the piston reaches thispredetermined position.

The device described in FIGS. 2 through 6 uses a piston and sealtraveling in a tapered tube, but a right circular cylinder may wellfunction adequately for mixtures of liquids other than blood and wherethe residual volume of the first liquid discharged is not too critical.The tapered tube has the advantages mentioned in the discussion offig.1. The position of the piston can be judged visually by the operatorrelative to graduations on the barrel (not shown), or an opticaldetector and automatic valve operation system can be used (not shown)

Since the residual enriched plasma is injected back into the patient thematerials used for this device have to be medical grade materials, atleast for those constituents contacting the blood. Polycarbonate or PTEare suitable for the barrel 206, end cap 215, sleeve 213, frame 208,knob 223 and collar 225. Insert 219 is of a suitable grade of passivatedstainless steel such as 416 or 420. The ball bearings have to do duty athigh speed but operate for very short times so stainless steel bearingsof grade ABMA 1-3 are adequate. O-rings 218 and seal 211 are of siliconerubber. Since the motor does not contact blood industrial motors (forexample those made by Mabucci) are adequate.

FIG. 7 shows an embodiment with a flexible bladder 312 that initiallyconforms to the bore of the barrel 306, the bladder providing a variablevolume chamber through its ability to invert as shown in FIGS. 10 and11. This embodiment may serve to reduce the effect of entrapped airbubbles.

In FIG. 7 a liquid tight variable volume centrifuge chamber (the BSC) isformed from a tapered barrel 306 containing a molded bladder 312, andend cap 315. The bladder is captured in a return fold 339 between abarrel projection 338 and the end cap 315. Larger end of barrel 306 isclosed by end cap 315. In the case where this device is used to enrichplasma from whole blood, end cap 315 has passages 316 and 317 boredwithin to permit the passage of red blood cells from passage 317 andplasma from passage 316. Passages 317 and 316 connect with valves formedby O-rings 318 compressed in recesses 326 operating in concert withports 328 and 327 respectively in sleeve 313. Sleeve 313 fits slidablyon end cap 315 to permit the ports 328 and 327 to connect with thepassages 316 and 317 at appropriate points in the operation. The knob323 and sleeve 313 can be placed in 3 positions: first position, port328 open and port 327 closed: second position, both ports 327 and 328closed: third position, port 328 closed and port 327 open. Sleeve 313 iskeyed to end cap 315 to permit the transmission of rotary motion betweenthese constituents (key not shown). Insert 319 is fastened to end cap315 to provide an axle for the ball bearing 320 supporting the left handend of the rotating assembly. Since the sleeve 313 is rotating with thechamber a ball bearing 321 is provide to connect the sleeve to anon-revolving knob 323 via collar 325 and rods 322. Barrel 306 isfastened to the shaft 305 of electric motor 301 using screw 307. Noadditional bearings are provided at the motor end, the motor bearingssufficing to support the barrel. The complete assembly is supported by aframe 308, the insert bearing 320 and the motor 301 being located onthis frame. The revolving components all rotate about axis XX. In thisillustration the sleeve is in the first position to keep the port 328open for porting of air as the chamber is charged with blood, and theplasma port 327 is closed. Whole blood 329 fully charges the chamber. Anelastomeric seal 314 permits the introduction of a needle 334 for thepassage of whole blood into the chamber before the start of rotation,and removal of enriched plasma at the cessation of action.

FIG. 8 is a transverse cross section of the device shown in FIG. 7 atsection AA. Whole blood 329 fills the BSC and bladder 312 which is fullyin contact with barrel 306. Frame 308 runs under the rotating assembly.

FIG. 9 shows the device of FIG. 7 in operation rotating at speed. Thesleeve 313 is in position two with both ports 327 and 328 closed. Theboundary between RBCs 331 and plasma 330 is shown at 337. The bladder isstill against the barrel now under the influence of the pressuredeveloped by the spinning liquid mixture.

FIG. 10 depicts the situation after spinning for 60 seconds or so. Thesleeve 313 is placed in position one, port 328 is open and RBCs 331 arebeing discharged through port 328. Plasma port 327 is closed. Thebladder has moved to the left to compensate for the volume of RBCs thathave been discharged. The shape adopted by the bladder is a balancebetween the forces developed by liquid pressure pushing the bladder tothe right and atmospheric pressure (via vent 332) pushing the bladder tothe left. Since the pressure at the center of the spinning liquid isnear absolute zero the atmospheric pressure exceeds the left handpressure that has been developed up to a certain radius, hence there-entrant shape of the bladder. The volume of plasma 330 has remainedthe same as when introduced. The boundary between RBCs and plasma isshown at 337. In this view the RBC discharge is about to be stoppedsince the residual RBC volume 331 is low enough.

FIG. 11 illustrates the final position for the bladder 312 while therotation continues but just prior to stopping. Sleeve 313 is in positionthree, RBC port 328 is closed and plasma port 327 is still open. Plasmahas been discharged through port 327 and is about to be cut off by thebladder rolling onto end cap 315 and cutting off the passage 316. Thisillustrates the minimum volume of enriched plasma 330. At this point thesleeve 313 is moved to position two with both ports closed and therotation is then stopped; the residual liquid is removed using a syringein a similar manner to the charging described in FIG. 7.

Materials for the device of FIGS. 7 through 11 are similar to those forthe device of FIGS. 2 through 6: the bladder by example can be made ofsilicone rubber, polyurethane or polyvinylchloride.

For the previous device 200 the piston position provided the signal forclosure of the RBC port 328. In the case of the bladder the invertedbladder rolls along the tapered barrel bore, the axial position of thereverse edge providing (labeled 312 in FIG. 11) the volume and thesignal for port closure. The cut-off of the plasma discharge isautomatic as the bladder rolls over the port passage 316.

The device described in FIGS. 12 through 16 utilizes an air core anduses no bladder or piston.

The device of FIG. 12 is very similar in construction to the twoprevious embodiments, with a BSC formed from a barrel 406 and end cap415. The inner surface of the end cap 415 forms the larger diameter endwall of the chamber, with the inner surface of the barrel 406 formingthe chamber's tapering side wall. In this illustration whole blood 429from syringe 433 fills the centrifuge chamber through needle 434 withboth ports 428 and 427 closed. Air displaced by the blood leaks outthrough the clearance between the needle 434 and insert 419 bore as theblood is injected. FIG. 13 shows the circular section nature of FIG. 12.Once the charging syringe is removed, the motor is started and thechamber is rotated at 10,000 to 20,000 rpm for approximately one minute.At this point the sleeve 413 is moved to the second position, and RBCsare discharged through port 428 until the point shown in FIG. 14 wherethe minimum RBCs 431 remain. Meanwhile, the plasma adopts the region orlayer 430, and a boundary 440 forms at the plasma-air radial interface,the air core 438 having entered through the bore of insert 419 (via afilter in the housing not shown, but see FIGS. 17 and 18). At thisjuncture the sleeve is moved to the third position, port 428 closed andport 427 opened. With this preferred device there is no bladder orpiston to observe, so the operator observes the axial interface 436between the RBCs 431 and the plasma 430 of the mixture through thetransparent barrel to determine when to manually close the RBC port 428and open the plasma port 427. With blood, this mixture interface is easyto see and can be automated with an optical detector. The difference inelectrical resistivity between red blood cells and plasma can also beused to trigger an indicator or automated valve. An alternative way ofdetermining the point at which to shut the RBC port is to use time.After one minute of running to separate the constituents of the blood,the RBC port is opened and a timer started. Since the pressure generatedin the centrifuge is a predictable function of liquid specific gravityand running speed, and since the RBC port is a precisely calibratedorifice, the flow rate being discharged, and hence time can be computedfor a given hematocrit value.

With the motor still running, the plasma discharges through port 427until it reaches the situation in FIG. 15 where the residual RBCs are atlayer 431 and the residual plasma at layer 430. The sleeve is then movedto the second position to close both ports. In the case of plasma thepassage 416 is placed at a precise radial location to give an accuratefinal volume since no further flow of plasma will occur once the aircore 438 has grown to that passage radial location. The motor is thenstopped and the device placed on end, with the motor downward, so thatthe rotation axis is vertical as shown in FIG. 16. The remainingenriched plasma with some RBCs is removed by syringe and needle asillustrated.

An enclosure suitable for all embodiments discussed in this applicationis described in FIGS. 17 and 18; however these two figures show theenclosure applied specifically to the air core embodiment of FIGS. 12through 16. The frame 508 is mounted to a battery power pack 503 thatacts as the base for the enclosure. An outer casing 500 surrounds thecentrifuge and is fastened to the battery pack 503, the joint beingliquid and air-tight. A valve selector knob 545, integral with eccentric546 and pin 547, is mounted in the casing such that the selector knob545 can be turned by the operator to actuate the internal knob 523 viathe pin 547 in groove 548 and hence the collar 525 and valve sleeve 513.In FIG. 17 the motor 501 driving the chamber BSC is controlled manuallyby switch 504 connected to battery pack 503 by wires 550. A bush 543mounted at the left hand end of the enclosure 500 provides alignment forthe entry of the syringe (433 FIG. 12) needle when charging the chamberwith whole blood or when extracting the enriched plasma. Immediatelyadjacent to bush 543 is a porous flexible pierceable filter 544. Thisfilter has two functions: It filters the air entering the core of thecentrifuge when it is running, and it prevents the egress of anyaerosols into the atmosphere of blood fragments generated as thecentrifuge discharges RBCs or plasma into the casing. A small slit inthe filter allows the charging syringe needle to enter without damagingthe effectiveness of the filter. Covering most of the interior walls ofthe casing 500 is a highly absorbent lining 542 to absorb the RBCS andplasma discharged into the casing as the air core 538 enlarges and theenrichment process proceeds. A lens and mask 549 placed in the wall ofthe casing 500 permits the operator to view the axial interface 536 ofthe RBCs and plasma as the process of enrichment proceeds. The mask andlens are chosen to enhance the contrast of the image seen of the liquidseparation interface 536.

A photo detector (not shown) can be placed in the location of the lensto provide an electrical signal of the progress of the liquid separationinterfaces, and an electromagnet actuator can drive the valve selectorknob 545. These electrical elements in conjunction with a manual switchcan be used to control the entire process once the motor has started.

From tests to date it would seem feasible in some applications to use asimple timer program to schedule the sleeve motions. For example, thefollowing sequence can operate off a timer once the chamber is chargedwith blood, a) start motor, run for 60 seconds b) open RBC port anddischarge RBCs for 30 seconds, c) close RBC port and open plasma portand run for 30 seconds, d) close both ports, and stop motor. Such adevice might require the addition of a means of manually inserting thepatient's hematocrit number to allow for varying proportions of RBCs toplasma.

Table 1 gives typical data obtained for the air core device of FIGS. 12through 16 using porcine blood. The data was obtained with runs of oneminute for the initial separation and approximately one more minute todischarge the RBCs and plasma.

TABLE 1 Platelet Count Platelet (×10³/ Concentration % Platelet % RedBlood Sample microliter) Factor Recovery Cells Removed Baseline 229 NANA NA Run 1 1656 7.2 100 93 Run 2 1457 6.4 88 92 Run 3 1446 6.3 87 93Run 4 1685 7.3 100 94

For all three embodiments discussed, piston, bladder and air core, thesize and position of the ports and passages are very important. As thecentrifuge rotates, the pressure developed within the chamber varies asthe square of the speed and the square of the radius of rotation. Togain manual control over the discharge of constituents the dischargeneeds to take place over a manageable time. The RBC port for exampleneeds to be sized to allow passage of the RBCs over a period of about 30seconds. Conditions must be selected to allow the RBC port to functionwithout blockage as the RBCs try to clump, and flow has to be kept lowenough to stop the platelets from being swirled into the exit vortex.For centrifuges using whole blood samples of approximately 30 mL, it hasbeen found that RBC ports of the order 0.008 inch diameter work well ifspeeds are in the region 15,000 to 20,000 rpm and chamber barrels areabout 1.0 to 1.25 inch in diameter at the largest point. Plasma portscan be larger since the risk of losing the platelets is less: values ofabout 0.010 inch diameter are adequate. Placement of the plasma portsrelative to the center axis of rotation has a direct effect on theattainable concentration factor. The closer to the center, the lessplasma is removed and less concentration is achievable. Additionally, inall embodiments of the invention discussed it will be noticed that asmall annulus 241, 341, 441, 541 is created at the large diameter end ofthe chamber. This annulus creates a localized area of increased radialdepth, but of small volume, for the RBCs prior to their entry into theRBC passages 217, 317, 417. This increase in depth reduces the tendencyfor the platelets and other desired factors from exiting with the RBCsbeing discharged through the RBC port 228, 328, 428 under influence ofthe exit vortex created locally close to the same ports (not shown).

In all the embodiments discussed the accuracy of the RBC port closurepoint can be improved by employing a flowable separator gel of anintermediate specific gravity between the red blood cells and theplatelets. The separator gel spreads over the red blood cell layermoving the other layers further towards the center axis. The separatorgel automatically caps the first port when all of the red blood cellshave exited. The separator gel viscosity is designed so that it will notpass through the small exit port at the centrifuge speed employed in theBSC. The automatic shut off of the first port can also be accomplishedwith a solid material of intermediate specific gravity that is designedto enter and close off the port when the red blood cells have fullyexited. An example would be plastic beads such as microspheres with thedesired intermediate specific gravity that are large enough to cap theport when agglomerated as they flow toward the port.

For the bladder and air core embodiments the visualization of the RBCplasma axial boundaries can be improved by incorporating back lightingin the form of an LED mounted inside the BSV adjacent to the motorcenterline. Additional windings in the motor could provide the low powerneeded to power the lamp.

With adjustments to size and locations of the port and passagedimensions, the subject invention also has the capability for separatingand concentrating a wide variety of therapeutically beneficial cells andother biological constituents. Many of these biological constituentshave the potential for regenerative therapy and can be characterized asregenerative agents. These regenerative agents can assist with theregeneration, restoration, or repair of a structure or assist with thefunction of an organ, tissue or physiologic unit or system to provide atherapeutic benefit to a living being. Examples of regenerative agentsinclude for example: stem cells, fat cells, progenitor cells, bonemarrow, synovial fluid, blood, endothelial cells, macrophages,fibroblasts, pericytes, smooth muscle cells, uni-potent and multi-potentprogenitor and precursor cells, lymphocytes, etc. The invention also hasthe potential to process soft or liquid tissues or tissue components ortissue mixtures including but not limited to adipose tissue, skin,muscle, etc. to provide a therapeutic regenerative agent.

The blood centrifuge container may also incorporate an adjustable port,e.g. a tube with an open end extending radially into the BSC and hingedat the outer periphery in such a manner that the tube can be swung in anarc for the open end to scan a range of radii (not shown). The locationof the open end of the tube can be adjusted before or during operationsuch that it is located at a desired position with respect to the axisof rotation. For example, the entrance port could be located towards theperiphery of the centrifuge container to initially vent undesired cells,and later adjusted towards the center of the container to vent plateletpoor plasma. Alternatively, if the plasma fraction is what is desired tobe removed, the port can be positioned so that essentially only plasmais tapped from the stratified mixture.

The apparatus may also be configured to shut off, or at least to ceaserotating, once a predetermined quantity of one or more constituents suchas plasma has been tapped. Specifically, a port may be positioned suchthat, upon stratification, the plasma constituent is adjacent the port.When the valve for that port is opened, plasma is dispatched out throughthe port. The port may also be configured with a sensor that senses thepresence or absence of plasma. As such, the apparatus can be configuredsuch that the barrel continues to rotate as long as plasma is sensed ator in the port, but when plasma is no longer sensed, the sensor providesa signal to the motor to stop (thereby stopping the rotation of thebarrel) or signaling the opening of a tap. As plasma continues to beremoved from the barrel through the port, eventually the supply ofplasma at the radius of the port is exhausted, thereby causing a signalto be sent from said sensor, and the barrel stops rotating. Of course,each of these signals may arise from the sensing of any stratifiedlayer, not just plasma.

It may be desirable to collect one or more of the discarded fractions ofthe liquid specimen in addition to the concentrated fraction. This canbe accomplished by one of several methods. A collection bag or chambercan be connected to an exit port on the sleeve. This bag or chamber willrotate with the barrel so provisions must be taken to balance it aroundthe axis of rotation. Another method would be to have a circumferentialfunnel opposite the desired exit port that would collect the fractionbeing discharged and guide the fluid to a collection point by gravityflow.

1-27. (canceled)
 28. A centrifuge for selectively concentrating andcollecting constituents of a biologic liquid mixture, said constituentshaving differing specific gravities and being stratifiable in acentrifugal field produced by said centrifuge, said centrifugecomprising: a) a chamber arranged to contain a liquid mixture and havinga central longitudinal axis about which said chamber is arranged to berotated to produce said centrifugal field, said chamber comprising: (i)an assembly comprising a tubular barrel and an end wall, each comprisinga common central longitudinal axis, said tubular barrel comprising aside wall tapering radially inward toward said central longitudinal axisfrom said end wall; (ii) an inlet for adding the liquid mixture to saidchamber; (iii) a first port in fluid communication with said chamber andlocated in said assembly at a first radial distance from said centrallongitudinal axis; and (iv) a second port in fluid communication withsaid chamber and located in said assembly at a second radial distancefrom said central longitudinal axis, said second radial distance beingsmaller than said first radial distance; b) a motor to rotate saidchamber about said central longitudinal axis to produce said centrifugalfield, whereupon said constituents of said biologic liquid mixture insaid chamber stratify into at least two concentric stratifiedconstituent layers as a function of the differing specific gravities ofsaid constituents, said at least two concentric stratified constituentlayers forming an interface between immediately adjacent constituentlayers thereof, and wherein a first of said at least two concentricstratified constituent layers is present at said first port, said firstport being selectively openable to enable at least a portion of saidfirst of said at least two concentric stratified constituent layers tobe automatically ejected from said chamber through said first port as aresult of pressure built up by said centrifugal field, said first portbeing arranged to be closed in response to a first signal, a second ofsaid at least two concentric stratified constituent layers being presentat said second port after closing of said first port, said second portbeing selectively openable to enable at least a portion of said secondof said at least two concentric stratified constituent layers to beautomatically ejected from said chamber through said second port as aresult of pressure built up by said centrifugal field; and c) a detectorfor detecting said interface and for providing said first signal inresponse thereto.
 29. The centrifuge of claim 28, additionallycomprising a vent to permit air to enter said chamber to at leastpartially replace a volume of said stratified constituent layer ejectedfrom said chamber.
 30. The centrifuge of claim 28, additionallycomprising a valve coupled to said first port and wherein said valve isarranged to open in response to said first signal.
 31. The centrifuge ofclaim 28, wherein said first signal is electrical or optical.
 32. Thecentrifuge of claim 28, whereupon the ejection of at least a portion ofsaid first of said at least two concentric stratified constituent layersout of said chamber through said first port leaves a residual portion ofsaid biologic liquid mixture in said chamber and wherein said chamberadditionally comprises another port for enabling the removal of at leasta portion of said residual portion of said biologic liquid mixture fromsaid chamber through said another port.
 33. The centrifuge of claim 28,wherein said detector detects said interface on the basis of colordifferences between said immediately adjacent stratified constituentlayers.
 34. The centrifuge of claim 28, wherein said detector detectssaid interface on the basis of differences in electrical conductivitybetween said immediately adjacent stratified constituent layers.
 35. Thecentrifuge of claim 28 wherein said first port is opened in automaticresponse said first signal.
 36. A method for selectively concentratingand collecting constituents of a biologic liquid mixture selected fromthe group consisting of blood, tissue or tissue components, saidconstituents having differing specific gravities and being stratifiablein a centrifugal field produced a centrifuge, said method comprising: a)providing a centrifuge having a chamber, an inlet, a first port and asecond port, said chamber having a central longitudinal axis andcomprising an assembly of a tubular barrel and an end wall arranged tocontain a liquid mixture, said first port being located a first radialdistance from said central longitudinal axis, said second port beinglocated a second radial distance from said central longitudinal axis,said second radial distance being smaller than said first radialdistance; b) introducing said biologic liquid mixture into said chamber;c) rotating said chamber about said central longitudinal axis to producesaid centrifugal field, whereupon said constituents of said liquidmixture in said chamber stratify into at least two concentric stratifiedconstituent layers as a function of the differing specific gravities ofsaid constituents and wherein a first one of said at least twoconcentric stratified constituent layers is present at said first port;d) selectively opening said first port to enable at least a portion ofsaid first one of said concentric stratified constituent layers to beautomatically ejected out of said chamber through said first port as aresult of pressure built up by said centrifugal field; e) closing saidfirst port upon the detection of a perceptible interface between saidfirst one of said at least two concentric stratified constituent layersand another of said at least two concentric stratified constituentlayers and while said first port is closed rotating said chamber aboutsaid central longitudinal axis to produce said centrifugal field,whereupon a second one of said at least two stratified constituentlayers is present at said second port; and f) selectively opening saidsecond port to enable at least a portion of said second one of said atleast two stratified constituent layers to be automatically ejected outof said chamber through said second port as a result of pressure builtup by said centrifugal field.
 37. The method of claim 36 wherein afterejection of said at least a portion of said second one of said at leasttwo stratified constituent layers a residual portion of said biologicliquid mixture remains in said chamber and wherein said methodadditionally comprises: g) withdrawing at least a portion of saidresidual portion of said biologic liquid mixture from said chamber toprovide a biologic agent.
 53. 38. The method of claim 36 wherein saidbiologic agent is provided to a living being for therapeutic purposes.39. The method of claim 38, wherein said biologic agent is provided to aliving being to assist with the regeneration, restoration or repair of astructure or assist with the function of an organ, tissue or physiologicunit or system of the living being to thereby provide a therapeuticbenefit to the living being.
 40. The method of claim 39 wherein saidbiologic liquid mixture is taken from a living to produce said biologicagent and wherein said biologic agent is provided back to the livingbeing.
 41. The method of claim 38, wherein said biologic agent isselected from the group consisting of stem cells, fat cells, progenitorcells, bone marrow, synovial fluid, blood, endothelial cells,macrophages, fibroblasts, pericytes, smooth muscle cells, unit-potentand multi-potent progenitor and precursor cells, and lymphocytes. 42.The method of claim 36 wherein said opening of said first and secondports is accomplished manually.
 43. The method of claim 36 wherein thedetection of said perceptible interface is accomplished visually. 44.The method of claim 43 wherein said chamber includes a transparentportion and wherein the detection of said perceptible interface isaccomplished visually through said transparent portion of said chamber.45. The method of claim 36 wherein said opening of said first and secondports is accomplished automatically.
 46. The method of claim 36, whereinsaid the detection of said perceptible interface is on the basis ofdifferences in electrical conductivity between said concentricstratified constituent layers.
 47. The method of claim 36 wherein saidbiologic liquid mixture comprises blood, wherein said stratifiedconstituent layer at said first port comprises red blood cells andwherein said stratified constituent layer at said second port comprisesplasma.
 48. The method of claim 47 wherein after ejection of said atleast a portion of said plasma some platelet concentrate remains in saidchamber and wherein said method additionally comprises: g) withdrawingat least a portion of said platelet concentrate from said chamber toprovide a biologic agent.
 49. The method of claim 48 wherein saidbiologic agent is provided to a living being to assist with theregeneration, restoration or repair of a structure or assist with thefunction of an organ, tissue or physiologic unit or system of the livingbeing to thereby provide a therapeutic benefit to the living being. 50.The method of claim 48 wherein said biologic liquid mixture is takenfrom a living to produce said biologic agent and wherein said biologicagent is provided back to the living being.